Conjugate

ABSTRACT

The present invention relates to a polypeptide comprising at least one alpha-helix having synthetically attached thereto a plurality of therapeutic or diagnostic moieties, wherein said therapeutic or diagnostic moieties may be the same or different and are spatially oriented on the polypeptide so as to minimise interactions between said moieties. Further aspects of the invention relate to a pharmaceutical composition comprising the polypeptide; a polynucleotide sequence encoding the polypeptide; an expression vector comprising said polynucleotide sequence; and a host cell transformed with said expression vector. The invention also provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of said polypeptide.

The present invention relates to the field of recombinant molecules thatare capable of delivering therapeutic agents to target cells.

BACKGROUND TO INVENTION

Current treatment of disease is predominantly non-targeted. Drugs areadministered systemically, e.g. orally, which exposes many other tissuesas well as the tissues which are diseased. In cancer therapy,chemotherapeutic drugs are specific for cells which are growing anddividing rapidly, as they work mainly by a mechanism which interfereswith DNA replication [1]. However, other cells may take up the drug andalso become intoxicated, such as rapidly dividing bone marrow stemcells, resulting in immunosupression. In infectious diseases,anti-bacterial drugs are introduced into the blood (orally or byinjection) and interfere with a particular bacterial metabolic pathway.Again, exposure to other tissues can result in side effects.Virally-infected cells are difficult to treat as their metabolism isnearly identical to uninfected cells.

It is widely acknowledged that the future of medicine lies in thetailoring of drugs to the disease. This means delivering the therapeuticagent to the correct target tissue, rather than the non-selective hitand miss approach of most of the conventional drug treatments usedtoday. This approach may result in the administration of lower doses,lower side effects and toxicities and overall better responses. Advancesin genomics may one day mean that drugs can be tailored to theindividual, as one individual's cancer may differ from another's.

There are many drugs used clinically today that are effective atdestroying or treating diseased cells, once they have accumulated in thecorrect tissue. The problem therefore lies with the specific targetingof drugs, rather than the effector mechanism. Examples of targetinginclude targeted ionising radiation as opposed to external beamradiotherapy [2], targeted chemotherapy drugs (e.g. methotrexate ordoxorubicin) as opposed to free drugs [3] and toxins [4]. Photodynamictherapy (PDT) is a particularly good example as it is already wellestablished in many treatments. However, it is becoming apparent that abetter therapeutic outcome may result from pre-targeting aphotosensitizing (PS) drug to the correct tissues in addition totargeting the light source, which is not accurate at a cellular level[5].

Targeting drugs or other effectors to the desired cells has beenpreviously proposed. One of the main approaches to targeting is to useantibodies as the targeting element of a multifunctional molecule [6].The ideal design for such a multifunctional molecule would be one whichis highly specific for diseased cells, able to carry many drugs withhigh capacity without compromising their function, and able to depositthe drug in the sub-cellular compartment which would primarily beaffected.

Antibody Targeting

Antibodies have naturally evolved to act as the first line of defence inthe mammalian immune system. They are complex glycoproteins which haveexquisite diversity and specificity. This diversity arises fromprogrammed gene shuffling and targeted mutagenesis, resulting inprobably a trillion different antibody sequences [7]. Consequently, thisdiversity means that antibodies can bind to practically any targetmolecule. It is now possible to mimic antibody selection and productionin vitro, selecting for recombinant human antibodies against virtuallyany desired target [8]. A significant number of biotechnological drugsin development are based on antibody targeting [6]. The most popular invitro selection technique is antibody phage display, where antibodiesare displayed and manipulated on the surface of viruses [8]. There aremany therapeutic antibodies being developed for a range of diseases,primarily cancer. Table 1 lists some of these antibodies.

Antibodies can bind with a high degree of specificity to target cellsexpressing the appropriate receptor. The affinity of an antibody is ameasure of how well an antibody binds to the target (antigen). It isusually described by an equilibrium dissociation constant (Kd).Technology exists to select and manipulate antibodies which have thedesired kinetic binding properties. For antibodies that need to beinternalised, the association rate is more important, as thedissociation rate does not function if the antibody is taken into thecell.

As with all biological molecules, the size of the antibody affects itspharmacokinetics in vivo [12]. Larger molecules persist longer in thecirculation due to slow clearance (large glycoproteins are clearedthrough specific uptake by the liver). For whole antibodies (molecularweight approx. 150 KDa) which recognise a cancer cell antigen in a mousemodel system, 30-40% can be taken up by the tumour, but because theypersist longer in the circulation, it takes 1-2 days for a tumour:bloodratio of more than one to be reached. Typical tumour:blood ratios are5-10 by about day 3 [13]. With smaller fragments of antibodies, whichhave been produced by in vitro techniques and recombinant DNAtechnology, the clearance from the circulation is faster (moleculessmaller that about 50 KDa are excreted through the kidneys, as well asthe liver). Single-chain Fvs (about 30 KDa) are artificial bindingmolecules derived from whole antibodies, but contain the minimal partrequired to recognise antigen [14]. Again in mouse model systems, scFvscan deliver 1-2% of the injected dose, but with tumour:blood ratiosbetter than 25:1, with some tumour:organ ratios even higher [15]. AsscFvs have only been developed over the last 10 years, there are notmany examples in late clinical trials. From clinical trials of wholeantibodies, the amount actually delivered to tumours is about 0.1 to 1%of that seen in mouse models, but with similar tumour:organ ratios [16].If another molecule is attached to the antibody, then the new sizedetermines the altered pharmacokinetic properties. Other properties suchas net charge and hydrophilicity have effects on the targeting kinetics[17].

Some cell surface antigens are static or very slowly internalise whenbound by a ligand such as an antibody. There are some which have afunction that requires internalisation, such as cell signalling oruptake of metals and lipids. Antibodies can be used to deliver agentsintracellularly. These agents can be therapeutic—repairing or destroyingdiseased cells. Examples include gene delivery [18], the intracellulardelivery of toxins (e.g. Pseudomonas exotoxin [4]), enzymes (e.g.ribonuclease [19]) and drugs (e.g. methotrexate [3]). Some of theseagents need targeting to particular sub-cellular organelles in order toexert their effects. Advances in cell biology have uncovered‘codes’—amino acid sequences which direct intracellular proteins tocertain sub-cellular compartments. There are specific sequences totarget to the nucleus, endoplasmic reticulum, golgi, lysosomes andmitochondria (Table 2).

There has been much research into targetable therapeutic drugs wherenovel effector functions have been linked to antibodies or othertargeting ligands. Some of these need to be internalised to successfullydeliver a toxic agent. Many of these have shown good results in vitroand in vivo in animal models, but have been disappointing in the clinic.Immunotoxins have shown problems such as immune reactions andliver/kidney toxicity [25]. There have been developments with new‘humanised’ immunotoxins based on enzymes such as ribonuclease [19] anddeoxyribonuclease [26]. These potentially have lower side effects andare more tolerable, but still do not have a bystander killing effect.Chemotherapy drugs tend to be much less active when linked to proteinsas they are not released effectively and radioimmunotherapy tends toirradiate other tissues en route to the tumour, giving rise to bonemarrow and liver toxicity. Photosensitising (PS) drugs are particularlyattractive agents to link to proteins, as the cytotoxic elements are thesinglet oxygen species generated from them and not the PS drugsthemselves [5].

Photodynamic Therapy (PDT)

Photodynamic therapy is a minimally invasive treatment for a range ofconditions where diseased cells and tissues need to be removed [27].Unlike ionising radiation, it can be administered repeatedly at the samesite. Its use in cancer treatment is attractive because conventionalmodalities such as chemotherapy, radiotherapy or surgery do not precludethe use of PDT and vice versa. Photodynamic therapy is also findingother applications where specific cell populations must be destroyed,such as blood vessels (in age-related macular degeneration (AMD) or incancer), the treatment of immune disorders, cardiovascular disease, andmicrobial infections. PDT is a two-step or binary process starting withthe administration of the PS drug, by intravenous injection, or topicalapplication for skin cancer. The physico-chemical nature of the drugcauses it to be preferentially taken up by cancer cells or other targetcells [28]. Once a favourable tumour (or other target):normal organratio is obtained, the second step is the activation of the PS drug witha specific dose of light, at a particular wavelength. This ultimatelycauses the conversion of molecular oxygen found in the cellularenvironment into reactive oxygen species (ROS) primarily singlet oxygen(¹O₂), although reactions of intermediate photochemically producedspecies also generate hydroxyl radicals (OH.) and superoxide (O₂ ⁻.).These molecular species cause damage to cellular components such as DNA,proteins and lipids [29]. PDT is a cold photochemical reaction, i.e. thelaser light used is not ionising and the PS drugs have very low systemictoxicity. The combination of PS drug and light result in low morbidityand insignificant functional disturbance and offers many advantages inthe treatment of diseases. There is growing evidence that PDT responserates and durability of responses are as good as or even superior tostandard locoregional therapies [27].

The light activation of ROS is highly cytotoxic. In fact some naturalprocesses in the immune system utilise ROS as a way of destroyingunwanted cells. These species have a short lifetime (<0.04 μs) and actover a short radius (<0.04 μm) from their point of origin. Thedestruction of cells leads to a necrotic area of tissue which eventuallysloughs away or is resorbed. The remaining tissue heals naturally,usually without scarring. There is no tissue heating and connectivetissue such as collagen and elastin are unaffected, resulting in lessrisk to the underlying structures compared to thermal laser techniques,surgery or external beam radiotherapy. More detailed research has shownthat PDT induces apoptosis (non-inflammatory cell death), and theresulting necrosis (inflammatory cell lysis) seen is due to the mass ofdying cells which are not cleared away by the immune system [30].

Generally PS drugs are administered systemically, with some topicalapplications for skin lesions. When the PS drug has accumulated in thetarget tissue, with ratios typically 2-5:1 compared with normalsurrounding tissues (except in the brain where the ratio can be up to50:1), low power light of a particular wavelength is directed onto thetumour (or the eye in AMD treatment [31]). Because human tissue cantransmit light most effectively in the red region of the visible lightspectrum, PS drugs which can absorb red light (630 nm or above) can beactivated up to a depth of about 1 cm. Patients must avoid sunlightuntil systemically administered PS drugs clear from the body, otherwisethey may have skin photosensitivity, resulting in skin burn.

The treatment scheme is attractive to the clinician in that superficialdiseases can usually be treated with local anaesthesia and sedation. Thegenerally low toxicity (with the possible exception of skinphotosensitivity) limits the need for other medication. Topicaltreatments do not require sterile conditions and can be given in anoutpatient clinic.

Research on a number of PS drugs including silicon phthalocyanines hasshown that PDT induces apoptosis-programmed cell death [32]. Apoptosisis the highly orchestrated and evolutionary conserved form of cell deathin which cells neatly commit suicide by chopping themselves intomembrane-packaged pieces [33]. These apoptotic bodies are marked forphagocytosis by the immune system. Usually, too much apoptosis in asmall area ‘overloads’ the immune system and the area eventually becomesnecrotic, with inflammatory consequences.

Photofrin (porfimer sodium), 5-aminolaevulanic acid (ALA) andVerteporfin (BPD-benzoporphyrin derivative) are three PS drugs whichhave regulatory approval. A promising, potent second generation PS drug,Foscan (temoporfin; meta-tetrahydroxyphenyl chlorin) is encounteringproblems in acquiring approval from the FDA and MCA. Porfimer sodium,the first PS drug to be approved, is licensed for use in bladder,stomach, oesophagus, cervix and lung cancer. Its performance is moderatedue to poor light absorption characteristics in the red end of thespectrum (activated at 630 nm), meaning it can only penetrate about 5 mminto tissues. It also persists in the body for weeks, leading to skinphotosensitivity. However it has been effective in the treatment of theabove cancers [27]. ALA is applied topically in the treatment of skinlesions and is converted endogenously to protoporphyrin IX, anaturally-occurring PS molecule. This can be activated at manywavelengths and its depth of effect is less than 2 mm. ‘Visudyne’(Verteporfin) also performs well in AMD [31], without the issues oftissue penetration found in tumour applications.

The newer generation of PS drugs have longer activation wavelengths thusallowing deeper tissue penetration by red light, higher quantum yieldand better pharmacokinetics in terms of tumour selectivity and residualskin photosensitivity. These classes of PS drugs include thephthalocyanines, chlorins, texaphyrins and purpurins. The syntheticchlorin, Foscan is a very potent PS drug with a wavelength of activationof 652 nm, good quantum yield of singlet oxygen and skinphotosensitivity of about 2 weeks. There have been many clinical trialsfor a variety of cancers, with good results [27]. There are other PSdrugs which have been developed and are in trials which can adsorb at740 nm, such as meso-tetrahydrophenyl bacteriochlorin (m-THPBC).

Clinical PDT

PDT can achieve disease control rates similar to conventional techniqueswith lower morbidity rates, simplicity of use and improved functionaland cosmetic outcome. PDT has mainly been used where conventionalapproaches have failed or are unsuitable. These include pre-malignantdysplastic lesions and non-invasive cancers which are commonly found inthe mucosa of aerodigestive and urinary tracts (e.g. oral cavity,oesophagus and bladder). Current treatments for cancer at this stage arenot very successful and good responses here would prevent larger solidtumours or metastatic spreads occurring. Treatment for Barrett'soesophagus usually involves an oesophagectomy, which requires generalanaesthesia, has a risk of morbidity and loss of function anddisfigurement. PDT is being seen as an attractive option because of thelarge area which can be treated superficially with less risk. Photofrin,ALA and Foscan have produced good responses in these types of cancers inclinical trials (Table 3).

Due to easy light accessibility, the treatment of cutaneous disease suchas skin cancer has produced good results with systemic and topical PSdrugs (Table 3). Head, neck and oral lesions have also produced goodresults and are well suited due to the good cosmetic outcome of thetreatment (Table 3). Treatment of other cancers are being tested asadvances are being made in laser and light delivery technology.Endoscopes can be used to deliver the activating light dose to anyhollow structure such as the oesophagus and bronchial cavity, thusexpanding the treatment range to gastrointestinal and lung cancers(Table 3) with minimal surgery. Large areas such as the pleura andperitoneum can be treated, where radiotherapy would not be able to givea high enough curative dose. PDT has great promise in the treatment ofthese surface serosal cancers, in combination with debulking surgery.Light can be delivered to these large surfaces in a short time, throughhollow cavities. The limited depth of activity would be an advantage, asthe critical underlying organs would be spared (Table 3). Adjuvanttherapy is also an option being investigated, where the solid tumour issurgically removed and any remaining tumour cells are destroyed by oneround of PDT in the cavity formed.

Although surface cancers may be the most amenable to PDT, solid tumoursmay also be able to undergo interstitial treatment, where the PS drug isadministered systemically or by intra-tumour injection, followed by theinsertion of laser fibres through needles equally spread throughout thetumour. This can result in necrosis of very large tumours (Table 3).

To summarise, there are several advantages of PDT therapy. It offersnon-invasive, low toxicity treatments which can be targeted by the lightactivation. The target cells cannot develop resistance to the cytotoxicspecies (ROS). Following treatment, little tissue scarring exists.However, PS drugs are not very selective for the target cells withtarget:blood ratios typically in single figures. Because PS drugs“piggy-back” on blood proteins, they persist longer in the circulationthan is desired, leaving the patient photosensitive for 2 weeks in thebest of cases. It is becoming increasingly clear that PS drugs need toaccumulate inside cells as the generated ROS have a short pathlength.This may not be achieved effectively with current PS drugs.

Targetable PDT

Photosensitiser drugs can still be active and functional while attachedto carriers, as the cytotoxic effect is a secondary effect resultingfrom light activation. This makes them very amenable to specific drugdelivery mechanisms. Currently, the approaches used to link PS drugs totargetable elements include direct conjugation of derivatised PS drugsto whole monoclonal antibodies or other ligands [34-37]. However, thisoften results in a heterogeneous mixture of antibody-PS drug moleculesas the chemistry is not accurate. Whole antibodies have a molecularweight of 150 KDa, resulting in very large immunoconjugates withunfavourable pharmacokinetics, such as poor tumour:organ ratios [36]which take a long time to achieve. It is also likely that PS drugslinked to large adjacent residues of a protein can have a detrimentaleffect on PS photophysics, with quenching of the desired PS excitedstates occurring due to adverse PS-protein interactions. Thenon-specific attachment of PS drugs onto antibodies or other ligands canresult in a severe compromise in binding ability of the ligand. Theantibody binding site may be hindered by such reactions, dramaticallylowering the affinity and specificity of the antibody. Too many PS drugsattached can also affect the hydrophobicity of a protein and may have anadverse effect on the structure and pharmacokinetics [36].

Some researchers have tried to circumvent these problems by attemptingto link PS drugs to designated ‘carriers’ such as chemically synthesisedbranched carbohydrate chains and poly-lysine chains. These approachesall require additional conjugation steps as the ligand-carriers cannotbe made entirely recombinantly. Using chains of pure poly-lysine mayalso give rise to problems, for example, proteolyic instability in vivo,or the concentration of hydrophobic PS drugs in one part of the moleculeleading to aggregation and quenching of adjacent PS drugs-excitedstates.

The present invention seeks to alleviate some of the above-mentionedproblems of the prior art, thereby providing an improved system fortargeting and delivery of therapeutic and/or diagnostic agents.

STATEMENT OF INVENTION

In a first aspect, the present invention provides a polypeptidecomprising at least one alpha-helix having synthetically attachedthereto a plurality of therapeutic or diagnostic moieties, wherein saidtherapeutic or diagnostic moieties may be the same or different and arespatially oriented on the polypeptide so as to minimise interactionsbetween said moieties.

A second aspect relates to the use of a polypeptide according to theinvention in the preparation of a medicament for the prevention and/ortreatment of disease.

A third aspect relates to a polynucleotide sequence encoding all or partof the polypeptide of the invention.

A fourth aspect relates to an expression vector comprising thepolynucleotide sequence of the invention.

A fifth aspect relates to a host cell transformed with the expressionvector or the polynucleotide sequence of the invention.

A sixth aspect relates to a method for preparing a polypeptide accordingto the invention comprising expressing the polynucleotide of theinvention, or culturing the host cell of the invention under conditionswhich provide for expression of the polypeptide.

A seventh aspect relates to a method of transporting a therapeutic ordiagnostic agent into a cell comprising exposing a cell to a polypeptideaccording to the invention.

An eighth aspect relates to a pharmaceutical composition comprising apolypeptide according to the invention and a pharmaceutically acceptablediluent, excipient or carrier.

A ninth aspect relates to a method of treatment comprising administeringto a subject in need thereof a therapeutically effective amount of apolypeptide according to the invention.

A tenth aspect relates to a diagnostic method comprising administeringto a subject a diagnostically effective amount of a polypeptideaccording to the invention.

An eleventh aspect relates to a method of preparing a polypeptideaccording to the invention, said method comprising conjugating atherapeutic or diagnostic agent to an alpha-helical polypeptide.

DETAILED DESCRIPTION

Various preferred features and embodiments of the invention aredescribed below.

As mentioned above, a first aspect of the present invention provides apolypeptide comprising at least one alpha-helix having syntheticallyattached thereto a plurality of therapeutic or diagnostic moieties,wherein said therapeutic or diagnostic moieties may be the same ordifferent and are spatially oriented on the polypeptide so as tominimise interactions between said moieties.

Preferably, said therapeutic or diagnostic moieties are spatiallyoriented on the polypeptide so as to minimise unfavourable or disruptiveinteractions between said moieties.

Typically, the polypeptide may be a conjugate, for example, a proteinconjugate, i.e., a fusion protein.

Preferably, the polypeptide of the invention comprises one or morespecific amino acid residues for the purpose of site-specificconjugation to said therapeutic or diagnostic moieties.

In one preferred embodiment, said specific amino acid residues compriseone or more basic amino acids.

In one preferred embodiment, said specific amino acid residues compriseone or more acidic amino acids.

In another preferred embodiment, said specific amino acid residuescomprise one or more hydroxyl-containing amino acids.

In another preferred embodiment, said specific amino acid residuescomprise one or more thiol-containing amino acids.

In another preferred embodiment, said specific amino acid residuescomprise one or more hydrophobic amino acids. By way of definition, theterm “hydrophobic amino acid residue” encompasses amino acids havingaliphatic side chains, for example, valine, leucine and isoleucine.

In a particularly preferred embodiment of the invention, the alpha-helixcomprises at least two functional amino acid residues positioned so asto protrude externally from said alpha-helix so that each functionalamino acid residue does not hinder another. Preferably, the functionalamino acid residues are suitable for cross-linking to one or moretherapeutic or diagnostic agents. Examples of such functional aminoacids include lysine, cysteine, threonine, serine, arginine, glutamate,aspartate, tyrosine.

Typically, the α-helix is proteolytically and temperature stable, and isdesigned so that functional groups from one type of side chain (e.g.basic residues such as lysine and arginine) protrude from the helix insuch a way that each functional group is spatially separated from eachother.

The length of the helical peptide may be varied to incorporate more orfewer functional amino acid residues, thereby accommodating more orfewer therapeutic agents respectively, as required. Likewise, theposition and number of functional amino acid residues can be altered toincrease or decrease the distance between the attached therapeuticagents, or to vary the number of therapeutic agents attached. In eachcase, the spatial arrangement of the functional amino acid residues issuch that there is little or no interference between the therapeuticagents attached thereto.

Preferably, the alpha-helix is a 19-residue helix with functional aminoacid residues at positions 2, 8, 10, 14 and 16.

By way of example, and as illustrated in FIG. 2A, the polypeptide maycomprise a 19-residue peptide helix with functional amino acids such aslysine or arginine residues at positions 2, 8, 10, 14, 16. This resultsin an approximately equal number of positively charged residuesabove/below or either side of the helical axis (viewed in FIG. 2B).These positively charged residues can be seen to be spatially separatedwhen the helix is viewed ‘end on’ (FIG. 2A).

Preferably, the side-chain type is the same for any one helix.

In one preferred embodiment, the polypeptide of the invention maycomprise two or more alpha-helical polypeptides in the form of amulti-helix bundle. Such multi-helix bundles enable the attachment of agreater number of therapeutic agents. Furthermore, without wishing to bebound by theory, it is believed that multi-helix bundles of this typemay exhibit an improved stability over the corresponding singlealpha-helical polypeptides.

In one preferred embodiment, the polypeptide of the invention comprisestwo alpha-helices, i.e., a two-helix bundle. These can be of asingle-chain or separate chain format.

In another preferred embodiment, the polypeptide of the inventioncomprises three alpha-helices, i.e., a three-helix bundle. Again, thesecan be of a single-chain or separate chain format.

In another preferred embodiment, the polypeptide of the inventioncomprises four alpha-helices, i.e., a four-helix bundle. Again, thesecan be of a single-chain or separate chain format. By way of example, afour-helix bundle is, shown in FIGS. 2C and 2D. This example shows a4-helix bundle with engineered cysteine residues for thiol coupling.Eight thiols are available for coupling which result in optimal spacingof the therapeutic agents when viewed from ‘end on’ (FIG. 2C) and fromthe side (FIG. 2D).

Preferably, the polypeptide of the invention comprises a natural orsynthetic four helix bundle.

In one especially preferred embodiment, the polypeptide of the inventionis the wild-type or mutant form of ‘rop’ (repressor of primer).

More preferably still, the polypeptide of the invention is a derivativeof wild-type or mutant form of ‘rop’ comprising cysteine or lysineresidues at optimal positions in the helix bundle.

Preferably, the polypeptide is scFv4-helix bundle-cys or scFv4-helixbundle-lys.

Targeting Element

In one particularly preferred embodiment of the invention, thepolypeptide further comprises a targeting element.

Preferably, the polypeptide of the invention is in the form of a fusionprotein. Thus, polypeptides according to the invention may includefusion proteins in which a targeting protein is linked to the alphahelix bearing a plurality of therapeutic or diagnostic agents via theirpolypeptide backbones through genetic expression of a DNA moleculeencoding these proteins, directly synthesised proteins, and coupledproteins in which pre-formed sequences are associated by a cross-linkingagent.

Preferably, the targeting element is selected from a recombinantantibody, a Fab fragment, a F(ab′)₂ fragment, a single chain Fv, adiabody, a disulfide linked Fv, a single antibody domain and a CDR.

As used herein, the term “CDR” or “complementary determining region”refers to the hypervariable regions of an antibody molecule, consistingof three loops from the heavy chain and three from the light chain, thattogether form the antigen-binding site.

By way of example, the antibody may be selected from Herceptin, Rituxan,Theragyn (Pemtumomab), Infliximab, Zenapex, Panorex, Vitaxin, Protovir,EGFR1 or MFE-23.

In one preferred embodiment, the targeting element is a geneticallyengineered fragment selected from a Fab fragment, a F(ab′)₂ fragment, asingle chain Fv, or any other antibody-derived format.

Conventionally, the term “Fab fragment” refers to a protein fragmentobtained (together with Fe and Fc′ fragments) by papain hydrolysis of animmunoglobulin molecule. It consists of one intact light chain linked bya disulfide bond to the N-terminal part of the contiguous heavy chain(the Fd fragment). Two Fab fragments are obtained from eachimmunoglobulin molecule, each fragment containing one binding site. Inthe context of the present invention, the Fab fragment may be preparedby gene expression of the relevant DNA sequences.

Conventionally, the term “F(ab′)₂” fragment refers to a protein fragmentobtained (together with the pFc′ fragment) by pepsin hydrolysis of animmunoglobulin molecule. It consists of that part of the immunoglobulinmolecule N-terminal to the site of pepsin attack and contains both Fabfragments held together by disulfide bonds in a short section of the Fcfragment (the hinge region). One F(ab′)₂ fragment is obtained from eachimmunoglobulin molecule; it contains two antigen binding sites, but notthe site for complement fixation. In the context of the presentinvention, the F(ab′)₂ fragment may be prepared by gene expression ofthe relevant DNA sequences.

As used herein, the term “Fv fragment” refers to the N-terminal part ofthe Fab fragment of an immunoglobulin molecule, consisting of thevariable portions of one light chain and one heavy chain. Single-chainFvs (about 30 KDa) are artificial binding molecules derived from wholeantibodies, but which contain the minimal part required to recogniseantigen.

In another preferred embodiment, the targeting element is a synthetic ornatural peptide, a growth factor, a hormone, a peptide ligand, acarbohydrate or a lipid.

The targeting element can be designed or selected from a combinatoriallibrary to bind with high affinity and specificity to the targetantigen. Typical affinities are in the 10⁻⁶ to 10⁻¹⁵ M K_(d) range.Functional amino acid residues, present in the targeting element, whichcould participate in the therapeutic agent attachment reaction may bealtered by site-directed mutagenesis where possible, without alteringthe properties of the targeting element. Examples of such changesinclude mutating any free surface thiol-containing residues (cysteine)to serines or alanines, altering lysines and arginines to asparaginesand histidines, and altering serines to alanines.

The target cells themselves can be human, other mammalian cells ormicrobial cells (e.g. anti-bacterial PDT using anti-bacterial antibodies[39]).

As discussed above the targeting element and the polypeptide may belinked directly or indirectly via a linker moiety. Direct linkage mayoccur through any convenient functional group on one of the proteins,such as a hydroxy, carboxy or amino group. Indirect linkage will occurthrough a linking moiety. Suitable linking moieties include bi- andmulti-functional alkyl, aryl, aralkyl or peptidic moieties, alkyl, arylor aralkyl aldehydes acids esters and anyhdrides, sulphydryl or carboxylgroups, such as maleimido benzoic acid derivatives, maleimido proprionicacid derivatives and succinimido derivatives or may be derived fromcyanuric bromide or chloride, carbonyldiimidazole, succinimidyl estersor sulphonic halides and the like. The functional groups on the linkermoiety used to form covalent bonds between the alpha helix and targetingelements may be two or more of, e.g., amino, hydrazino, hydroxyl, thiol,maleimido, carbonyl, and carboxyl groups, etc. The linker moiety mayinclude a short sequence of from 1 to 4 amino acid residues thatoptionally includes a cysteine residue through which the linker moietybonds to the transport protein. Alternatively, the targeting element andthe polypeptide may be linked by leucine zippers, dimerisation domains,or an avidin/biotin linker.

Additional Sequences

In an especially preferred embodiment of the invention, the polypeptidefurther comprises one or more additional sequences selected from asub-cellular targeting peptide and a membrane active peptide. Theadditional amino acid sequence may be attached either to the targetingelement or to the alpha helix of the polypeptide, or to both.

Examples of sub-cellular targeting peptides include nuclear localisationsequences (NLS), mitochondrial localisation sequences, lysosomaltargeting peptides, endoplasmic reticulum retrieval signals, golgitargeting sequences. These sequences serve to deliver the therapeuticagent to certain subcellular compartments, particularly the nucleus. Theadditional sequences can also be membrane-active peptides (Table 2)which function to disrupt the endosomal compartment containing thefusion protein after internalisation. This will facilitate the releaseof the therapeutic agent into the cytosol of the cell where it can havea potent action.

In one particularly preferred embodiment, the sub-cellular targetingpeptide targets the nucleus and comprises a sequence selected fromKKKKRPR and KRPMNAFIVWSRDQRRK.

In another particularly preferred embodiment, the sub-cellular targetingpeptide targets the mitochondria and comprises the sequenceMLVHLFRVGIRGGPFP GRLLPPLRFQTFSAVRYSDGYRSSSLLRAVAHLPSQLWA.

In yet another particularly preferred embodiment, sub-cellular targetingpeptide targets lysosomes and comprises the sequence KCPL.

In another particularly preferred embodiment, the sub-cellular targetingpeptide allows proteins to traffic back to the endoplasmic reticulum andcomprises the sequence KDEL.

In one especially preferred embodiment, the membrane active peptidetargets the membrane and comprises a sequence selected from thefollowing: (i) GLFGAIAGFIENGWEGMIDGWYG; (ii) GIEDLISEVAQGALTLVP; (iii)ACYCRIPACIAGERRYGTCIYQGRLWAFCC; and (iv) FFGAVIGTIALGVATSAQITAGIALAEAR.Glycosylated Peptides

In one preferred embodiment, the polypeptide comprises a protein havingone or more N- or O-linked carbohydrate residues spatially oriented soas to minimise interactions between said carbohydrates or therapeutic ordiagnostic moieties attached thereto.

Thus, in one preferred embodiment, the polypeptide comprises aglycosylated protein e.g. human serum albumin) or comprises a proteinhaving one or more N- or O-linked glycosylation sites. By way ofdefinition, the term “glycosylated protein” refers to a glycoprotein,i.e., a protein having one or more carbohydrates attached thereto.Typically, glycoproteins contain oligosaccharide units linked to eitherasparagine side chains by N-glycosidic bonds, or to serine and threonineside chains by O-glycosidic bonds. Accordingly, a protein having N- orO-linked glycosylation sites includes any protein containing amino acidresidues having one or more OH or NH₂ side chains.

These proteins may be expressed in a eukaryotic system such as mammaliancells, yeasts or insect cells, to ensure full glycosylation. Derivatisedtherapeutic agents, whose chemistry is compatible with chemicalattachment to hydroxyl or carboxylate groups may be cross-linked ontothe glycosylated proteins. The types of carbohydrate residues found onglycosylated proteins are shown in FIG. 1.

In another preferred embodiment of the invention, the polypeptidecomprises one or more glycosylation motifs. Typical examples of suchglycosylation motifs include Asn-X-Ser and Asn-X-Thr, wherein X is anyamino acid residue. Polypeptide sequences including these glycosylationmotifs may be expressed in eukaryotic hosts, for example, yeast. Methodsfor expressing polypeptide sequences may be accomplished by standardprocedures well known to those skilled in the art.

After glycosylation, therapeutic and/or diagnostic agents may beattached to the carbohydrate residues by standard chemical techniques.The spatial arrangement of the glycosylation motifs is such that thereis little or no interference between the therapeutic or diagnosticagents attached thereto.

Therapeutic and Diagnostic Agents

As mentioned above, the polypeptide of the invention has a plurality oftherapeutic or diagnostic agents synthetically attached thereto. As usedherein, the term “therapeutic agent” refers to any therapeutic agentcapable of giving rise to a therapeutic effect, either directly orindirectly.

As used herein, the term “synthetically attached” encompassesstraightforward chemical synthetic techniques and also in vivo synthesisusing recombinant DNA techniques. The term is not intended to encompassnaturally occurring molecules.

By way of example, typical diagnostic agents include fluorescentporphyrins (for use in microscopy or sub-cellular localisation studies[40]), palladium or platinum-based porphyrins (for use in oxygen-sensingapplications, or linked to antibodies against relevant biologicalmarkers), paramagnetic or radiolabelled porphyrins (for use in imagingstudies), or gadolinium-porphyrins (for use as contrast agents).

The therapeutic or diagnostic agent may be attached directly to thepolypeptide, or by virtue of a linker group. Direct linkage may occurthrough any convenient functional group on one of the proteins, such asa hydroxy, carboxy or amino group. Indirect linkage may occur through alinking moiety, for example, those suitable for linking the alpha helixand targeting elements, as described hereinafter in the preparationsection.

Preferably, the therapeutic agent is a chemotherapeutic agent or ananti-infectious agent. Examples of chemotherapetuic agents includemethotrexate and doxorubicin, whilst examples of anti-infectious agentsinclude metronidazole, nisin, curvacin, netilmicin, amikacin, MicrocinB17, rifabutin and sparfloxacin.

In an alternative preferred embodiment, the therapeutic agent is atherapeutic peptide or protein. Typical examples may include peptidesthat are toxic or corrective, natural or synthetic.

In yet another preferred embodiment, the therapeutic agent is a nucleicacid. Nucleic acids can be attached for the purposes of corrective ordestructive cell gene therapy.

The therapeutic agent may also be a boronated porphyrin (for boroncapture neutron therapy-BCNT [38].

More preferably, the therapeutic agent is a photosensitising agent.Typical photosensitising agents may include, for example,meta-tetrahydroxyphenyl chorin, 5-aminolaevulanic acid,BPD-benzoporphyrin derivative, meso-tetrahydrophenyl bacteriochlorin,chlorin e₆, pyropheophorbide-a, bacteriochlorin-a and sulfonatedaluminium phthalocyanine.

By linking novel or established PS agents to small, targetable carrierproteins specifically designed to accept these PS drugs withoutcompromising their function, the invention allows delivery of a highlyspecific dose of PS drug to a target tissue, which can later beactivated by light. These carrier-PS drug conjugates are advantageousover existing targeted and non-targeted PDT approaches in that a greateramount of the PS agent can accumulate in the target tissue, often withtissue to blood/normal organ ratios of 100:1 or better, in shorter timeintervals. These agents also have advantages over other targetablestrategies as they give rise to lower side effects and result in littleor no immunogenicity. The type and number of PS agents attached can becontrolled very accurately by engineering the polypeptide molecules,thus obtaining optimal physical and biological characteristics. The factthat the toxic species is generated in the second step means that theagent is not toxic during the delivery step and the toxic species doesnot have to be released from the polypeptide.

In a particularly preferred embodiment, the polypeptide of the inventionis used in combination with one or more inhibitors of one or more oxygenradical scavenging enzymes. The use of such inhibitors effectivelyincreases the availability of reactive oxygen species (ROS) in thetarget tissue. Inhibitors of this type may be administeredconsecutively, simultaneously or sequentially with the polypeptide ofthe invention. Alternatively, said one or more inhibitors may beattached by virtue of a linker moiety to the polypeptide, for example,by attaching to any of the above-mentioned functional amino acidresidues. Typical examples of suitable enzyme inhibitors include3-amino-1,2,4-triazole (catalase inhibitor), taxifolin(glutathione-S-transferase inhibitor), mercaptosuccinate (glutathioneperoxidase inhibitor) and 2-methoxyoestradiol (superoxide dismutaseinhibitor).

Derivatised therapeutic agents, such as photosensitiser drugs, whosechemical properties are compatible with chemical linking to aminegroups, can be attached to the helix using standard chemical techniques.This results in a helix carrying therapeutic agents which are optimallyseparated so that there is little or no interference between each drugmolecule. Such interference may be in the form of chemical quenching,photo-chemical quenching or steric hindrance.

Therapeutic and Diagnostic Applications

A further aspect of the invention relates to the use of a polypeptide asdescribed above in the preparation of a medicament for the preventionand/or treatment of disease. As used herein the phrase “preparation of amedicament” includes the use of a polypeptide of the invention directlyas the medicament in addition to its use in a screening programme forthe identification of further agents or in any stage of the manufactureof such a medicament. Diseases which may be treated according to theinvention include cancer, age-related macular degeneration, microbialinfections, arthritis and other immune disorders and cardiovasculardisease.

Another aspect relates to a method of treatment comprising administeringto a subject in need thereof a therapeutically effective amount of apolypeptide according to the invention.

Likewise, yet another aspect relates to a diagnostic method comprisingadministering to a subject a diagnostically effective amount of apolypeptide according to the invention.

Yet another aspect of the invention relates to a method of transportinga therapeutic or diagnostic agent into a cell comprising exposing a cellto a polypeptide according to the invention.

Polynucleotide Sequences

Another aspect of the invention provides a polynucleotide sequenceencoding all or part of the polypeptide of the invention.

As used herein the term “polynucleotide” refers to a polymeric form ofnucleotides of at least 10 bases in length and up to 1,000 bases or evenmore, either ribonucleotides or deoxyribonucleotides or a modified formof either type of nucleotide. The term includes single and doublestranded forms of DNA.

Polynucleotides may be constructed using standard recombinant DNAmethodologies. The nucleic acid may be RNA or DNA. Where it is RNA,manipulations may be performed via cDNA intermediates. Reference may bemade to Molecular Cloning by Sambrook et al. (Cold Spring Harbor, 1989)or similar standard reference books for exact details of the appropriatetechniques.

Sources of nucleic acid may be ascertained by reference to publishedliterature or databanks such as GenBank. Nucleic acid encoding thedesired polypeptide sequences may be obtained from academic orcommercial sources where such sources are willing to provide thematerial or by synthesising or cloning the appropriate sequence whereonly the sequence data are available. Generally this may be done byreference to literature sources which describe the cloning of the genein question.

Alternatively, where limited sequence data is available or where it isdesired to express a nucleic acid homologous or otherwise related to aknown nucleic acid, exemplary nucleic acids can be characterised asthose nucleotide sequences which hybridise to the nucleic acid sequencesknown in the art.

It will be understood by a skilled person that numerous differentnucleotide sequences can encode the same peptides used in the presentinvention as a result of the degeneracy of the genetic code. Inaddition, it is to be understood that skilled person may, using routinetechniques, make nucleotide substitutions that do not affect thepeptides encoded by the nucleotide sequence of the present invention toreflect the codon usage of any particular host organism in which thepeptide of the present invention is to be expressed.

Variants/Homologues/Derivatives

In addition to the specific amino acid sequences and nucleotidesequences mentioned herein, the present invention also encompasses theuse of variants, homologues and derivatives thereof. Here, the term“homologue” means an entity having a certain homology with the subjectamino acid sequences and the subject nucleotide sequences. Here, theterm “homology” can be equated with “identity”.

With respect to sequence homology, preferably there is at least 75%,more preferably at least 85%, more preferably at least 90% homology tothe reference sequences. More preferably there is at least 95%, morepreferably at least 98%, homology. Nucleotide homology comparisons maybe conducted using a sequence comparison program such as the GCGWisconsin Bestfit program.

Typically, the homologues will comprise the same sequences that code forthe active sites etc. as the subject sequence. Although homology canalso be considered in terms of similarity (i.e. amino acid residueshaving similar chemical properties/functions), in the context of thepresent invention it is preferred to express homology in terms ofsequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the. default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However, for some applications, it is preferred to usethe GCG Bestfit program. A new tool, called BLAST 2 Sequences is alsoavailable for comparing protein and nucleotide sequence (see FEMSMicrobiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1):187-8 and tatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M NQ Polar - charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) may occur i.e. like-for-like substitution such as basic forbasic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Replacements may also be made by unnatural amino acids include; alpha*and alpha-disubstituted* amino acids, N-alkyl amino acids*, lacticacid*, halide derivatives of natural amino acids such astrifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*,p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyricacid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-aminocaproic acid^(#), 7-amino heptanoic acid*, L-methionine sulfone^(#*),L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*,L-hydroxyproline^(#), L-thioproline*, methyl derivatives ofphenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)^(#), L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionicacid^(#) and L-Phe (4-benzyl)*. The notation * has been utilised for thepurpose of the discussion above (relating to homologous ornon-homologous substitution), to indicate the hydrophobic nature of thederivative whereas # has been utilised to indicate the hydrophilicnature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than theα-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences for use in the present invention may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences described herein may bemodified by any method available in the art. Such modifications may becarried out in to enhance the in vivo activity or life span ofnucleotide sequences of the present invention.

Preparation, Expression Vectors and Host Cells

The polypeptides of the present invention may be prepared by any methodknown in the art, including recombinant DNA techniques. Alternatively,the polypeptide may be a naturally occurring polypeptide.

The present invention also relates to vectors which comprise apolynucleotide useful in the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof peptides useful in the present invention by such techniques.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or polynucleotides of the invention.Introduction of a polynucleotide into the host cell can be effected bymethods described in many standard laboratory manuals, such as Sambrooket al, such as calcium phosphate or chloride transfection, DEAE-dextranmediated transfection, transvection, microinjection, cationiclipid-mediated transfection, electroporation, transduction, scrapeloading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, NSO, HeLa, C127, 3T3, BHK, 293 and Bowesmelanoma cells; and plant cells.

A great variety of expression systems can be used to produce apolypeptide useful in the present invention. Such vectors include, amongothers, chromosomal, episomal and virus-derived vectors, e.g., vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand/or purification.

Chemically coupled sequences can be prepared from individual proteinsequences and coupled using known chemically coupling techniques. Thepolypeptide can be assembled using conventional solution- or solid-phasepeptide synthesis methods, affording a fully protected precursor withonly the terminal amino group in deprotected reactive form. Thisfunction can then be reacted directly with a second protein or asuitable reactive derivative thereof. Alternatively, the amino group maybe converted into a different functional group suitable for reactionwith a second protein. Thus, e.g. reaction of the amino group withsuccinic anhydride will provide a selectively addressable carboxylgroup, while further peptide chain extension with a cysteine derivativewill result in a selectively addressable thiol group. Once a suitableselectively addressable functional group has been obtained in thedelivery vector precursor, a second protein may be attached through e.g.amide, ester, or disulphide bond formation. Cross-linking reagents whichcan be utilized are discussed, for example, in Neans, G. E. and Feeney,R. E., Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43.

The present invention also provides a method of preparing a polypeptideas described above, said method comprising conjugating a therapeutic ordiagnostic agent to an alpha-helical polypeptide.

In a preferred embodiment, the method further comprise the step ofplacing the polypeptide so prepared in a container for subsequenttherapeutic or diagnostic use.

Preferably, the container has attached thereto a label indicatingregulatory approval for said therapeutic or diagnostic application.

Pharmaceutical Compositions

A further aspect of the invention provides a pharmaceutical compositioncomprising a polypeptide as described hereinbefore and apharmaceutically acceptable diluent, excipient or carrier (includingcombinations thereof).

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine and will typically comprise any one ormore of a pharmaceutically acceptable diluent, carrier, or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examplesof suitable diluents include ethanol, glycerol and water.

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like.

Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to beadministered using a mini-pump or by a mucosal route, for example, as anasal spray or aerosol for inhalation or ingestable solution, orparenterally in which the composition is formulated by an injectableform, for delivery, by, for example, an intravenous, intramuscular orsubcutaneous route. Alternatively, the formulation may be designed to beadministered by a number of routes.

Where the composition is to be administered mucosally through thegastrointestinal mucosa, it should be able to remain stable duringtransit though the gastrointestinal tract; for example, it should beresistant to proteolytic degradation, stable at acid pH and resistant tothe detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution which may containother substances, for example enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration the compositions may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

The present invention will now be described by way of example and withreference to the following figures wherein:

FIG. 1 shows the modular structure of the multifunctionaltargetable-carrier protein of the invention.

FIG. 2 shows the molecular structure of helical based carrier proteinsfor therapeutic agents. In more detail, FIGS. 2(A) and (B) show a singlepeptide α-helix engineered to contain optimally-spaced lysine orarginine residues, which can be used to deliver PS or other drugs. Side(B) and end-on (A) views show favourable spacing of the amino groupsused to attach the drugs. FIGS. 2(C) and (D) show a 4-helix bundle,engineered to contain optimally-spaced cysteine residues, which can beused to deliver PS or other drugs. Side (B) and end-on (A) views showfavourable spacing of the thiol groups used to attach the drugs.

FIG. 3 shows the construction of the scFv-4-helix bundle fusion gene. Inmore detail, FIG. 3 shows how a scFv and a 4-helix bundle gene would beassembled in a bacterial expression vector to produce the scFv-helixbundle fusion protein.

FIG. 4 shows over-expression anti-CEA scFv (lanes 5-7) and scFv-4 helixbundle (lanes 1-4) fusion protein in E. coli BL21(DE3). (A) Whole celllysates are analysed by SDS-PAGE stained with coomassie blue. (B) Wholecell lysates are analysed by western blot using a mouse anti-His tagmonoclonal antibody (Qiagen) followed by anti mouse-horseradishperoxidase (Sigma) developed by ECL (Amersham). M-molecular weightmarkers in KDa. Lane 8 represents substantially pure scFv-4 helix bundlefusion protein after IMAC on Nickel sepharose.

FIG. 5 shows antigen binding ELISA of scFv-4 helix bundle. In moredetail, a dilution series of scFv-4 helix bundle coupled to chlorin e₆is added to CEA immobilised on a microtitre plate and binding isvisualised using a mouse anti-His tag monoclonal antibody (Qiagen)followed by anti mouse-horseradish peroxidase (Sigma) developed usingo-phenyldiamine (OPD) substrate.

FIG. 6 shows the absorbance spectrum of a scFv-4 helix bundle fusionprotein coupled to chlorin e₆ sensitiser, compared to free sensitiser.The spectrum was measured using a UV-Vis spectrometer in a 1 cm cuvette.

FIG. 7 shows cell killing activity of a scFv-4 helix bundle fusionprotein coupled to clorin e₆ sensitiser. Cell killing was analysed onCEA-antigen positive cells (LS174T). Various concentrations of scFv-4helix bundle fusion protein-chlorin e₆ conjugate and free chlorin e₆ wasadded and cell cytotoxicity was measured after 2 hours binding andexposure to light. The light energy dose was 2J using an LED source of600-700 nm. Cell death was measured using the Cytotox-96 kit (Promega).Preliminary results indicate that the targeted PDT is about 10-fold moreeffective than the free sensitiser.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J.E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A:Synthesis and Physical Analysis of DNA Methods in Enzymology, AcademicPress. Each of these general texts is herein incorporated by reference.

EXAMPLES Example 1 1.1 Synthesis and Utility of scFv-4 Helix BundleFusion Protein Carrying PS Drug Molecules

A chosen, well characterised scFv is PCR amplified and cloned as an NcoI/Not I fragment into the bacterial expression vector pET20b (Novagen)to create pETscFv. A DNA cassette containing a 4 helix bundle (e.g. aderivative of the bacterial protein ‘rop’) is PCR amplified and clonedinto the Not I site of pETscFv to create pETscFv4HB (FIG. 3).Appropriate DNA primers are used introduce cysteine residues at optimalpositions in the helix bundle and to replace any cysteine residues inthe scFv (with residues which do not significantly alter the bindingcharacteristics of the scFv, such as serine, alanine and glycine). Theresulting construct is called pETscFv4HBcys

The vector pETscFv4HBcys is transformed into E. coli BL21(DE3) (Novagen)by the calcium chloride method [41] and plated onto 2TY agar platescontaining 100 μg/ml ampicillin [41]. Single colony transformants arepicked and re-streaked onto fresh 2TY Agar plates containingamplicillin.

A single colony is picked and grown in 5 ml of 2TY media containing 100μg/ml ampicillin at 30° C., in a shaking incubator (250 rpm) for 8-16hr. This culture is then used to inoculate a culture of 500 ml 2TY mediacontaining 100 μg/ml ampicillin and grown under similar conditions for afurther 3-16 hr.

The culture supernatant is harvested and concentrated using an Amiconultrafiltration stirred cell with a 30 KDa cut-off membrane to a finalvolume of 10 ml. Alternatively, the bacterial periplasm can be preparedusing the sucrose osmotic shock method [19] in a volume of 10 ml.

The concentrated supernatant or periplasmic extract is dialysed for 16hr against 5 L of phosphate-buffered saline (PBS) containing 0.5 M NaCland 2 mM MgCl₂. This is then applied to a copper (II) or nickel(II)-charged chelating sepharose column (Amersham-Pharmacia Biotech) andpurified by immobilised metal affinity chromatography (IMAC) for exampleas described in Deonarain et al [19]. The recombinant fusion proteinshould elute in an imidazole gradient at between 40 and 150 mMimidazole. The eluted fusion protein is further purified by gelfiltration on a superdex-200 column (Amersham-Pharmacia Biotech)equilibrated in PBS. FIG. 4 shows shows data for the expression andpurification of the resulting fusion protein, scFv-4-helix bundle-cys.

1.2 Coupling of Porphyrins to scFv-4-helix Bundle-cys

A porphyrin (5-(3-aminophenyl)-10,15,20-triphenylporphyrin isderivatized by reacting with excess bromoacetyl-bromide (dissolved inacetone containing sodium carbonate) to form(5-(3-bromoacetamidophenyl)-10,15,20-triphenylporphyrin. 10 μmol of thederivatized porphyrin and 10 mg of the scFv-4-helix bundle-cys aredissolved separately in 20 ml of dry dimethyl formamide. These twosolutions are added over a period of 3 hours to 50 ml dry dimethylformamide containing 25 mg sodium carbonate. These reactions areperformed in the dark, under argon. The final product is scFv-4-helixbundle-cys with optimally conjugated porphyrins (scFv-4-helixbundle-cys/porphyrin). The scFv-4-helix bundle-cys/porphyrin is dialysedinto PBS and concentrated as above.

The number of porphyrins attached to the scFv-4-helix bundle-cys fusionprotein is determined using electrospray mass spectrometry, compared tothe scFv-4-helix bundle-cys alone. To confirm the position of attachmenton the 4-helix bundle, the protein will be fragmented by trypsindigestion and the resulting peptides analysed by mass spectrometry.

1.3 Photophysical Studies

The photophysical characteristics of the scFv-4-helixbundle-cys/porphyrin are measured and compared to the free porphyrin.This includes UV/Visible absorption spectrum, fluorescence spectrum,fluorescence decay times, triplet state spectrum, singlet oxygen yieldand quenching experiments of triplet state by substrates.

1.4 Binding Studies

In vitro binding characteristics of the scFv-4-helixbundle-cys/porphyrin molecule are carried out by ELISA [42] or byBIACore surface plasmon resonance using published methods, compared tothe unmodified scFv and scFv-4-helix bundle. Cell binding of thescFv-4-helix bundle-cys/porphyrin can also be compared to the unmodifiedproteins can be determined by Fluorescently Activated Cell Sorting(FACS), confocal fluorescence microscopy.

1.5 Cytotoxicity Studies

In vitro cell cytotoxicity is measured as follows: The target cells (inthis example, an antigen positive cell line such as MCF7) are seeded ata concentration of 1×10⁴ cells per well in a 96-well microtitre plate inDMEM media/10% FCS. Cells are allowed to grow overnight in a humidifiedincubator at 37° C., with 5% CO₂. The next day, concentrationsscFv-4-helix bundle-cys/porphyrin ranging from 1 μM to 1 nM are added tothe wells in triplicate. After 3-6 hours, the media is washed 3 times incomplete media and the cells are exposed to light from a 500 W halogenlamp for 10-20 mm. The total light dose is about 25-50 J/cm². Thefollowing day, cell death is measured by lactase dehydrogenase release(Cytotox-96 kit, Promega). These experiments are done on an antigen cellline (e.g. KB cells). The molecules tested are the unmodified scFv, thescFv-4-helix bundle, free porphyrin and scFv-4-helixbundle-cys/porphyrin.

In vivo tumour eradication can be demonstrated as follows: Approx 1×10⁶MCF7 cells are injected s.c. into the flank of a nude BALB/C mouse andtumours are allowed to establish for 1-2 weeks. 50-500 μg ofscFv-4-helix bundle-cys/porphyrin is injected intravenously into thetail vein of tumour-bearing mice and allowed to accumulate in the tumourover a period of 12-36 hrs. At a time when the tumour:normal organ ratiois high (10:1 or better), light is irradiated onto the tumours. The sizeof the tumours is measured using callipers and compared to mice carryingantigen-negative tumours, and in animals injected with scFv alone,scFv-4-helix bundle, free porphyrin and scFv-4-helixbundle-cys/porphyrin.

Example 2

2.1 Preparation of pETscFv4HBLys

A scFv-4 helix bundle was prepared in accordance with the methodologydescribed in Example 1 and FIGS. 1 to 3, except that appropriate primerswere used to introduce lysine residues at optimal positions in the helixbundle. The resulting construct is called pETscFv4HBLys. An scFv whichtargets CEA (carcinoembryonic antigen) was used.

FIG. 4 shows the expression of four such clones in E. coli BL21(DE3)compared with the scFv (against CEA) alone. The expression is generallygood with yields of pure protein of approx. 1 mg/L cell culture.

2.2 Coupling of Chlorin e₆ to scFv4-helix Bundle-Lys

The N-hydroxysuccinimide (NHS) ester of the photosensitiser chlorin e₆,was prepared by reacting 1.5 equivalents of dicyclohexylcarbodiimide and1.5 equivalents of NHS with one equivalent of chlorin e₆ in dry dimethylsulphoxide (DMSO). The reaction was carried out under an inert gas (e.g.argon) and in the dark at room temperature and was complete in 2 hours,(tlc: silica gel 3% methanol in chloroform). A similar procedure can beused to prepare the active esters of other carboxyl containingphotosensitisers.

N-ethylmorpholine (1 μl), DMSO (10 ml) and the scFv-4 helix bundle (100μg in approx. 1 ml of PBS buffer) were stirred together under nitrogenat room temperature. To this solution was added the DMSO solutioncontaining the photosensitiser-NHS ester. The solution was stirred atroom temperature in the dark for 12 hours to synthesise the bundlechlorin e₆ conjugate. The conjugate was then dialysed against 2×5 L ofPBS. All procedures were carried out in the dark.

FIG. 5 shows target binding data for the chlorin e₆-scFv4-helixbundle-Lys conjugate, as measured by the technique described above inparagraph 1.4. FIG. 6 shows photophysical data for the resulting chlorine₆-scFv4-helix bundle-Lys conjugate measured in accordance withparagraph 1.3 above. FIG. 7 shows cell killing activity using cell lineLS174T, as measured by the technique described above in paragraph 1.5.

Various modifications and variations of the described methods of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in the relevant fields are intended to be covered by thepresent invention.

References

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[42] Harlow, E. & Lane, D. (1999). Using Antibodies. A LaboratoryManual. Cold Spring Harbor. TABLE 1 Therapeutic uses of Antibodies [29]Antibody Target Application Herceptin ErbB2 (Her 2) receptor Breastcancer therapy Rituxan CD20 Lymphoma Theragyn (Pemtumomab) Muc-1 Ovariancancer [30] Infliximab TNFα Rheumatoid arthritis, Crohn's diseaseZenapax CD25 Allograft rejection Panorex 17-1A surface antigenColorectal cancer Vitaxin αVβ3 intergrin Sarcoma ProtovirCytomegalovirus CMV infection (CMV) MFE-23 Carcinoembryonic Colorectalcancer [31] antigen

TABLE 2 Peptide sequences which could be used for sub-cellularlocalisation Name of Sequence Function Amino Acid Sequence [ref.] SV 40large T Targets polypeptides to KKKKRPR [20] nuclear localisation thenucleus Human SRY Targets polypeptides to KRPMNAFIVWSRDQRRK the nucleus[20] ATP-binding protein N- Targets polypeptides to MLVHLFRVGIRGGPFPGRLterminal peptide containing the mitochondria LPPLRFQTFSAVRYSDGYRmitochondria targeting SSSLLRAVAHLPSQLWA [21] Lysosomal membrane Targetspolypeptides to KCPL [22] targeting the lysosomes Endoplasmic reticulumAllows proteins to traffic KDEL [23] (ER) Retention Signal back to theER Influenza Haemaglutinin Disrupts membrane GLFGAIAGFIENGWEGMID HA2GWYG [24] Polio virus vp1 Disrupts membrane GIEDLISEVAQGALTLVP [24]Human defensin Disrupts membrane ACYCRIPACIAGERRYGTCI YQGRLWAFCC [24]Sendai virus fusion protein Disrupts membrane FFGAVIGTIALGVATSAQIT F1AGIALAEAR [24]

TABLE 3 Clinical Results with PDT in cancer [27] Disease PhotosensitiserResult Barrett's mucosal cancer Porfimer sodium 75% conversion to normalepithelium and tumours eliminated Barrett's oesophagus Systemic ALAHigh-grade dysplasia cancer eradicated in all patients Bladder cancerHematoporphyrin 74% complete response, derivative 30% alive after 5years Basal cell cancer of skin Topical ALA 90% complete response Oralcancer Dihematoporphyrin 87% complete response ether over 5-53 monthsChest wall recurrence in Dihematoporphyrin 20% complete response, breastcancer ether 45% partial response

1. A polypeptide comprising at least one alpha-helix havingsynthetically attached thereto a plurality of therapeutic or diagnosticmoieties, wherein said therapeutic or diagnostic moieties may be thesame or different and are spatially oriented on the polypeptide so as tominimise interactions between said moieties.
 2. A polypeptide accordingto claim 1 which comprises one or more specific amino acid residues forthe purpose of site-specific conjugation to said therapeutic ordiagnostic moieties.
 3. A polypeptide according to claim 2 wherein saidspecific amino acid residues comprise one or more basic amino acids. 4.A polypeptide according to claim 2 wherein said specific amino acidresidues comprise one or more acidic amino acids.
 5. A polypeptideaccording to claim 2 wherein said specific amino acid residues compriseone or more hydroxyl-containing amino acids.
 6. A polypeptide accordingto claim 2 wherein said specific amino acid residues comprise one ormore thiol-containing amino acids.
 7. A polypeptide according to claim 2wherein said specific amino acid residues comprise one or morehydrophobic amino acids.
 8. A polypeptide according to claim 1 whereinsaid alpha-helix comprises at least two functional amino acid residuespositioned so as to protrude externally from said alpha-helix so thateach functional amino acid residue does not hinder another.
 9. Apolypeptide according to claim 1 wherein said alpha-helix is a 19residue helix with functional amino acid residues at positions 2, 8, 10,14 and
 16. 10. A polypeptide according to claim 1 which comprises twoalpha-helices.
 11. A polypeptide according to claim 1 which comprisesthree alpha-helices.
 12. A polypeptide according to claim 1 whichcomprises four alpha-helices.
 13. A polypeptide according to claim 1which comprises a natural or synthetic four helix bundle.
 14. Apolypeptide according to claim 1 wherein said polypeptide is thewild-type or mutant form of ‘rop’ (repressor of primer).
 15. Apolypeptide according to claim 1 which further comprises a targetingelement.
 16. A polypeptide according to claim 15 wherein said targetingelement is selected from a recombinant antibody, a Fab fragment, aF(ab′)₂ fragment, a single chain Fv, a diabody, a disulfide linked Fv, asingle antibody domain and a CDR.
 17. A polypeptide according to claim15 wherein said targeting element is a synthetic or natural peptide, agrowth factor, a hormone, a peptide ligand, a carbohydrate or a lipid.18. A polypeptide according to claim 1 which further comprises one ormore additional amino acid sequences selected from a sub-cellulartargeting peptide and a membrane active peptide.
 19. A polypeptideaccording to claim 18 wherein said sub-cellular targeting peptidetargets the nucleus and comprises a sequence selected from KKKKRPR(SEQIDNO:1) and KRPMNAFIVWSRDQRRK (SEQIDNO:2).
 20. A polypeptideaccording to claim 18 wherein said sub-cellular targeting peptidetargets the mitochondria and comprises the sequence MLVHLFRVGIRGGPFPGRLLPPLRFQTFSAVRYSDGYRSSSLLRAVAHLPSQLWA (SEQIDNO:3).
 21. 2 LApolypeptide according to claim 18 wherein said sub-cellular targetingpeptide targets lysosomes and comprises the sequence KCPL (SEQIDNO:4).22. A polypeptide according to claim 18 wherein said sub-cellulartargeting peptide allows proteins to traffic back to the endoplasmicreticulum and comprises the sequence KDEL (SEQIDNO:5).
 23. A polypeptideaccording to claim 18 wherein said membrane active peptide targets themembrane and comprises a sequence selected from the following: (i)GLFGAIAGFIENGWEGMIDGWYQ; (SEQ ID NO:6) (ii) GIEDLISEVAQGALTLVP; (SEQ IDNO:7) (iii) ACYCRIPACIAGERRYGTCIYQGRLWAFCC; (SEQ ID NO:8) and (iv)FFGAVIGTIALGVATSAQITAGIALAEAR. (SEQ ID NO:9)


24. (A polypeptide according to claim 1 wherein said polypeptidecomprises a glycosylated protein.
 25. A polypeptide according to claim 1wherein said polypeptide comprises a protein having one or more N- orO-linked carbohydrate residues spatially oriented so as to minimiseinteractions between said carbohydrates or therapeutic or diagnosticmoieties attached thereto.
 26. A polypeptide according to claim 1wherein said therapeutic agent is a chemotherapeutic agent or ananti-infectious agent.
 27. A polypeptide according to claim 1 whereinsaid therapeutic agent is a photosensitising agent.
 28. A polypeptideaccording to claim 27 wherein said photosensitising agent is selectedfrom meta-tetrahydroxyphenyl chorin, 5-aminolaevulanic acid,BPD-benzoporphyrin derivative, meso-tetrahydrophenyl bacteriochlorin,chlorin e₆, pyropheophorbide-a, bacteriochlorin-a and sulfonatedaluminium phthalocyanine.
 29. A polypeptide according to claim 1 whereinsaid therapeutic agent is a therapeutic peptide or protein.
 30. Apolypeptide according to claim 1 wherein said therapeutic agent is anucleic acid.
 31. Use of a polypeptide according to claim 1 in thepreparation of a medicament for the prevention and/or treatment ofdisease.
 32. A polynucleotide sequence encoding all or part of thepolypeptide of claim
 1. 33. An expression vector comprising thepolynucleotide sequence of claim
 32. 34. A host cell transformed withthe expression vector of claim
 33. 35. A method for preparing apolypeptide according to claim 1 comprising expressing thepolynucleotide of claim
 32. 36. A method of transporting a therapeuticor diagnostic agent into a cell comprising exposing a cell to apolypeptide according to claim
 1. 37. A pharmaceutical compositioncomprising a polypeptide according claim 1 and a pharmaceuticallyacceptable diluent, excipient or carrier.
 38. A method of treatmentcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a polypeptide according to claim
 1. 39. A diagnosticmethod comprising administering to a subject a diagnostically effectiveamount of a polypeptide according to claim
 1. 40. A method of preparinga polypeptide according to claim 1, said method comprising conjugating atherapeutic or diagnostic agent to an alpha-helical polypeptide.
 41. Amethod according to claim 40 which further comprise the step of placingthe polypeptide so prepared in a container for subsequent therapeutic ordiagnostic use.
 42. A method according to claim 41 wherein saidcontainer has attached thereto a label indicating regulatory approvalfor said therapeutic or diagnostic application.
 43. A host celltransformed with the polynucleotide sequence of claim
 32. 44. A methodfor preparing a polypeptide according to claim 1 comprising culturingthe host cell of claim 34 under conditions which provide for expressionof the polypeptide.